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Espina G.,University of Bath | Eley K.,TMO Renewables Ltd | Pompidor G.,German Electron Synchrotron | Schneider T.R.,German Electron Synchrotron | And 2 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2014

Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding a G. thermoglucosidasius β-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed in Escherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6Å) structure of the enzyme-substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of the G. thermoglucosidasius β-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity. © 2014 International Union of Crystallography.

Extance J.,University of Bath | Crennell S.J.,University of Bath | Eley K.,TMO Renewables Ltd | Cripps R.,TMO Renewables Ltd | And 2 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD+ pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.514;Å resolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or 'spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms. © 2013 International Union of Crystallography Printed in Singapore - all rights reserved. © 2013.

Van Zyl L.J.,University of Cape Town | Taylor M.P.,University of Cape Town | Taylor M.P.,TMO Renewables Ltd | Eley K.,TMO Renewables Ltd | And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2014

This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75∈×∈105 M-1 s-1 at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35∈±∈0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling. © 2013 Springer-Verlag Berlin Heidelberg.

Taylor M.P.,TMO Renewables Ltd. | Taylor M.P.,University of the Western Cape | Mulako I.,University of the Western Cape | Tuffin M.,University of the Western Cape | Cowan D.,University of the Western Cape
Biotechnology Journal | Year: 2012

Alcohol-based liquid fuels feature significantly in the political and social agendas of many countries, seeking energy sustainability. It is certain that ethanol will be the entry point for many sustainable processes. Conventional ethanol production using maize- and sugarcane-based carbohydrates with Saccharomyces cerevisiae is well established, while lignocellulose-based processes are receiving growing interest despite posing greater technical and scientific challenges. A significant challenge that arises from the chemical hydrolysis of lignocellulose is the generation of toxic compounds in parallel with the release of sugars. These compounds, collectively termed pre-treatment inhibitors, impair metabolic functionality and growth. Their removal, pre-fermentation or their abatement, via milder hydrolysis, are currently uneconomic options. It is widely acknowledged that a more cost effective strategy is to develop resistant process strains. Here we describe and classify common inhibitors and describe in detail the reported physiological responses that occur in second-generation strains, which include engineered yeast and mesophilic and thermophilic prokaryotes. It is suggested that a thorough understanding of tolerance to common pre-treatment inhibitors should be a major focus in ongoing strain engineering. This review is a useful resource for future metabolic engineering strategies. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

van Zyl L.J.,University of the Western Cape | Taylor M.P.,TMO Renewables Ltd | Trindade M.,University of the Western Cape
Applied Microbiology and Biotechnology | Year: 2016

Geobacillus thermoglucosidasius is a promising platform organism for the production of biofuels and other metabolites of interest. G. thermoglucosidasius fermentations could be subject to bacteriophage-related failure and financial loss. We develop two strains resistant to a recently described G. thermoglucosidasius-infecting phage GVE3. The phage-encoded immunity gene, imm, was overexpressed in the host leading to phage resistance. A phage-resistant mutant was isolated following expression of a putative anti-repressor-like protein and phage challenge. A point mutation was identified in the polysaccharide pyruvyl transferase, csaB. A double crossover knockout mutation of csaB confirmed its role in the phage resistance phenotype. These resistance mechanisms appear to prevent phage DNA injection and/or lysogenic conversion rather than just reducing efficiency of plating, as no phage DNA could be detected in resistant bacteria challenged with GVE3 and no plaques observed even at high phage titers. Not only do the strains developed here shed light on the biological relationship between the GVE3 phage and its host, they could be employed by those looking to make use of this organism for metabolite production, with reduced occurrence of GVE3-related failure. © 2015, Springer-Verlag Berlin Heidelberg.

Bartosiak-Jentys J.,Imperial College London | Eley K.,TMO Renewables Ltd. | Leak D.J.,Imperial College London
Applied and Environmental Microbiology | Year: 2012

The pheB gene from Geobacillus stearothermophilus DSM6285 has been exploited as a reporter gene for Geobacillus spp. The gene product, catechol 2,3-dioxygenase (C23O), catalyzes the formation of 2-hydroxymuconic semialdehyde, which can be readily assayed. The reporter was used to examine expression from the ldh promoter associated with fermentative metabolism. © 2012, American Society for Microbiology.

Cooper G.R.,University of Sheffield | Cooper G.R.,TMO Renewables Ltd. | Moir A.,University of Sheffield
Journal of Bacteriology | Year: 2011

The paradigm gerA operon is required for endospore germination in response to L-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for L-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-aminoacid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore. © 2011, American Society for Microbiology.

TMO Renewables Ltd | Date: 2013-03-13

A thermophilic micro-organism comprising a modification that increases amylase expression and starch hydrolysis compared to wild-type, wherein the modification is insertion of a heterologous amylase gene.

A thermophilic microorganism comprising a modification that prevents sporulation, wherein the modification inactivates the native spo0A gene

TMO RENEWABLES Ltd | Date: 2012-01-25

A thermophilic micro-organism comprising a modification that increases amylase expression and starch hydrolysis compared to wild-type, wherein the modification is insertion of a heterologous amylase gene.

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